1. Field of the Invention
The present invention in the fields of molecular biology, biochemistry and medicine relates to novel protein domains involved in cytoskeletal events that are useful as a research tool and as inhibitors of cell growth, inducers of apoptosis and anticancer therapeutics.
2. Description of the Background Art
The recently discovered formin homology (FH) protein family participate in a range of actin-mediated processes affecting cell polarity and shape, including the in spatial and temporal coordination of cytokinesis. Substantial evidence indicates that FH proteins fulfil these functions by interacting, through distinct domains, with the actin-binding proteins profilin as well as the Arp2/3 complex and GTP-binding proteins of the Rho family.
The FH proteins were defined on the basis of conservation in sequence and protein organization among two Drosophila melanogaster proteins, DIAPHANOUS (DIA) and CAPPUCCINO (CAPU), a yeast protein, Bni1p, and a mouse protein, formin. Subsequently, additional genes encoding FH proteins have been found in fungi, plants, worms and mammals; there are now nearly a dozen characterized family members (Table 1). FH proteins are 1000 to 2000 amino acids in length and contain two conserved sequence domains, termed FH1 and FH2. FH1 domains average ˜100 amino acids in length, and most are extremely proline rich, with multiple stretches of consecutive proline residues. FH2 domains are conserved regions of ˜130 residues found only among members of the FH protein family.
The FH1 and FH2 domains in all family members are separated by ˜160 aa, whereas the lengths of the flanking regions vary considerably. Blocks of sequence similarity within these variably sized regions have become recognizable as the number of known family members has increased. Sequence conservation has also been detected in regions surrounding the FH2 domain.
All of the FH proteins other than CAPU contain one or more coiled-coil regions, as predicted by the NEWCOILS and PAIRCOILS algorithms. Typically, one coiled-coil region lies N-terminal to the FH1 domain, and one or two lie within or C-terminal to the FH2 region. Coiled-coil domains are common among cytoskeletal proteins and provide the potential for homotypic and heterotypic interactions.
Database searches with a consensus FH2 domain indicate that eukaryotes contain multiple FH genes (Table 1). In Saccharomyces cerevisiae, for which a complete genome sequence is available, there are two recognizable FH genes (BNI1 and BNR1), whereas there are at least three in other species (e.g., sequences U40187, U88314 and Z78013 in Caenorhabditis elegans). Although the two FH genes in S. cerevisiae overlap in function, the same does not appear to be true for the pairs of genetically identified FH genes in flies and fission yeast.
TABLE 1FH PROTEIN FAMILY MEMBERSCellularPairingAccessionProteinSpeciesfunctionspartnersano.Bni1pS. cerevisiaeCytokinesis;Profilin,Z71547cellRho1p,polarityCdc42p,Bud6p/Alp3pBnr1pS. cerevisiaeCytokinesis;Profilin, Rho4pZ47047cellpolaritycdc12S. pombeCytokinesisProfilinZ68136fus1S. pombeCellL37838polaritySepAA. nidulansCytokinesis;U83658cellpolarityA. thalianaZ97338CYK1C. elegansCytokinesisU40187CAPUD. melanogasterCellProfilinU34258polarityDIAD. melanogasterCytokinesisProfilinU11288inDiaM. musculusProfilin, RhoU96963hDIAH. sapiensForminM. musculusWW motifs, SH3X62379domainsaEndogenous interactions with the pairing partners listed have been confirmed only for the yeast FH proteins.bThis Table is reproduced Table 1 of Wasserman, Trends in Cell Biology 8:111-115, 1998; cited reference numbers are those listed in the Wasserman publication.Abbreviations: A. nidulans, Aspergillus nidulans; A. thaliana, Arabidopsis thaliana; CAPU, CAPPUCCINO; C. elegans, Caenorhabditis elegans; DIA, DIAPHANOUS; D. melanogaster, Drosophila melanogaster; FH, formin homology; H. sapiens, Homo sapiens; M. musculus, Mus musculus; S. cerevisiae, Saccharomyces cerevisiae; S. pombe, Schizosaccharomyces pombe; SH3, Srchomology 3.
The Diaphanousrelated formins (DRFs) constitute a subclass of FH proteins known for their ability to bind activated Rho subfamily of small GTP-binding proteins (Wasserman, S. Trends in Cell Biol, 1998, 8:111-115). Members of the Rho subfamily of GTP-binding proteins link FH proteins to cellular signalling pathways. Rho proteins, Ras-related GTPases, regulate cell adhesion, motility, bud-site selection and contractile processes (Takai, Y. et al. (1995) Trends Biochem. Sci. 20, 227-231; Narumiya, S. (1996) J. Biochem. 120, 215-228). The Rho subfamily includes the Rho, Rac and Cdc42 proteins, of which both Rho and Cdc42 are required for cytokinesis. It is these two proteins that interact with members of the FH family.
Ridley and Hall first demonstrated in 1992 that Rho small GTPase activation was both necessary and sufficient for the formation of actin stress fibers. Ridley, A. J. et al., Cell 70, 389-99 (1992a). Since then, two Rho GTPase bindimg proteins, Rho-kinase, or ROCK, and the Diaphanous-related Formin Homology proteins (DRFs) have been shown to be critical effectors in Rho-regulated actin remodeling (Ridley, A. J. Nat Cell Biol 1, E64-6 (1999)). Co-expression of activated variants of these two effectors are sufficient to recapitulate actin stress fibers caused by expression of activated RhoA (Kohno, H. et al., EMBO J 15, 6060-8 (1996); Watanabe, N. et al., Nat. Cell Biol. 1, 136-143 (1999); Nakano, K. et al., Mol Cell 5, 13-25 (2000); Tominaga, T. et al., Mol Cell 5, 13-25 (2000)). While ROCK has multiple substrates that participate in cytoskeletal remodeling, such as LIM kinase and the myosin-binding subunit (MBS) of myosin phosphatase (reviewed in Amano, M. et al., Exp Cell Res 261, 44-51 (2000)), none of the known DRF binding partners, including Src, IRSp53 and the actin-binding proteins, profilin, EF1α or Bud6p/Aip3p, appear to have a direct role in DRF-controlled actin remodeling (Watanabe, Y. et al., Mol Cell Biol 17, 2615-23 (1997); Umikawa, M. et al., Oncogene 16, 2011-6 (1998); Fujiwara, T. et al., Biochem. Biophys. Res. Comm. 271, 626-629 (2000); Ozaki-Kuroda, K. et al., Mol Cell Biol 21, 827-39 (2001); Suetsugu, S. et al., Embo J 17, 6516-26 (1998)). Instead, these factors likely have an obligatory role in targeting the Rho-regulated DRF complex to specific cellular regions or modulate DRF effects on the cytoskeleton.
In the budding yeast Saccharomyces cerevisiae, the DRFs include Bni1 and Bnr1 Evangelista, M. et al. (1997) Science 276, 118-122; Kohno, H. et al. (1996) EMBO J. 15, 6060-6068). Three mammalian DRF genes have been identified in mice/humans, respectively: mDia1/aNA1 Watanabe, N. et al. (1999) Nature Cell Biol. 1:136-143; Lynch, E. D. et al. (1997) Science 278, 1315-1318, mDia2/Dia2 (Alberts, A. S. et al., Cell 92, 475-487 (1998)), and mDia3/DIA (Bione, S. et al., Am J Hum Genet 62, 533-541 (1998)). mDia1 has been shown to bind activated RhoA-C and mDia2 binds RhoA, B and Cdc42 (Watanabe, N. et al., supra; Watanabe, N. et al. (1997) EMBO J. 16, 3044-3056). Based on primary amino acid sequence homology, the DRF family contains four conserved domains: the GTPase-binding domain (GBD) in the amino-terminus, three FH domains, the proline-rich FH1, FH2, and the FH3 domains (Petersen, J. et al., J Cell Biol 141, 1217-1228 (1998); Castrillon, D. H. et al. (1994) Development 120, 3367-3377). The present inventor has identified a new homology domain unique to the DRFs that termed the DRF-autoregulatory domain (DAD) in the extreme carboxyterminus that is described herein.
Studies in budding yeast showed that the DRFs are critical Rho effectors (Kohno et al., supra; Evangelista et al., supra). In mammalian cells, inhibition of the DRFs by microinjected antibodies showed that the DRFs are required for cytokinesis, stress fiber formation and activation of the transcription factor SRF (Tominaga, T et al., (2000) Molec. Cell 5:13-25). Expression of deregulated or ‘activated’ DRFs, created by removal of their GBD's, is sufficient to induce actin polymerization and gene expression in the absence of extracellular stimulation [Tominaga et al., supra; Watanabe. et al. (1999) supra; Evangelista et al., supra). These activated mouse DRFs also cooperate with another small GTPase effector, Rhokinase or ROCK, to induce stress fibers in fibroblasts (Nakano, K. et al., Mol Biol Cell 10, 2481-2491 (1999); Tominaga et al., supra; Watanabe, N. et al., supra.).
The DRFs act as adaptor molecules in cells and bridge signaling and remodeling pathways by binding to several signaling kinases and scaffolding proteins via SH3 domain interactions with the proline-rich FH1 domain. These include Src non-receptor tyrosine kinase family (Kikyo, M. et al. Oncogene 18, 7046-7054 (1999)), Hoflp (Fujiwara, T. et al., Biochem. Biophys. Res. Comm. 271, 626-629 (2000)), DIP-1 (Chang, F. et al., J Cell Biol 137, 169-182 (1997)), and IRSp53/BAIAP2 (Watanabe et al., 1997, supra). The actin binding protein profilin also interacts with FH1 domains (Imamura, H. et al. Embo J 16, 2745-2755 (1997); Tominaga, T. et al. supra; Narumiya, S., et al., FEBS Lett 410, 68-72 (1997); Mikawa, M. et al., Oncogene 16, 2011-2016 (1998)). Other actin binding factors EF1α and Bud6p/Aip3p associate with Bni1p through other regions (Evangelista, M. et al., supra; Suetsugu, S., et al., FEBS Lett 457, 470-474 (1999)). The significance of profilin binding to the mammalian DRF family members has yet to be elucidated, as it does not appear to be an important factor in Rho-regulated actin remodeling (Sotiropoulos, A. et al., Cell 98, 159-169 (1999)). It may act, however, as an actin-monomer sensor in an SRF regulatory pathway (Kikyo, M. et al., supra; Nakano, K. et al., supra).
Bni1p, mDia1 and mDia2 have been shown to be ‘activated’ or deregulated by removal of their GTPase binding domains. Expression of □GBD-mDia1 and -mDia2 in fibroblasts activates SRF in the absence of extracellular stimulation in addition to cooperating with another small GTPase effector, Rho-kinase or ROCK, to induce stress fibers ((Watanabe, N. et al., 1999, supra; Kikyo et al., supra; Zhao, Z. S. et al., Mol Cell Biol 18, 2153-2163 (1998)). These truncation experiments suggested that the GTPase binding domain of the DRFs contained a negative regulatory activity. Many signaling molecules contain autoregulatory domains. For example, p21-activated kinase (PAK1)
Frost, J. A. et al., J Biol Chem 273, 28191-28198 (1998); Burbelo, P. D. et al., J Biol Chem 270, 29071-29074 (1995)) and Src-family kinases bear domains that modulate their activity through intramolecular associations (Kim, A. S. et al., Nature 404, 151-158 (2000)). The PAK1 autoinhibitory domain is adjacent to the CRIB domain (Welch, M. D., Trends Cell Biol 9, 423-427 (1999)); it is presumed that this association is regulated by binding to activated Cdc42.
A similar observation has been made for the Cdc42-binding Wiskott-Aldrich syndrome protein (WASP) (Alberts et al., supra). WASP is activated by the Rho-related small GTPase Cdc42 (Rohatgi, R. et al., Cell 97, 221-31 (1999); Ma, L. et al., Proc Natl Acad Sci USA 95, 15362-7 (1998); Kim, A. S. et al., Nature 404, 151-8 (2000)), the binding of which disrupts an intramolecular association between the GTPase binding domain (CRIB) (Burbelo, P. D. et al., J Biol Chem 270, 29071-4 (1995)) and the C-terminal WCA domain. Free WCA then binds actin monomers and activates Arp2/3 actin remodeling complex to induce filopodia, lamellipodia and rufile formation (Machesky, L. M. et al., J Cell Biol 146, 267-72 (1999); Mullins, R. D. et al., Curr Opin Cell Biol 12, 91-6 (2000)). The Arp2/3 complex is composed of p16, p20, p21, p34, p41, Arp2 and Arp3 (using the vertebrate nomenclature as per Mullins, R. D. et al., Curr Opin Struct Biol 9, 244-9 (1999)). Arp2/3 regulates the assembly of actin filaments at the leading edges of cells by both promoting the nucleation of new actin fibers as well as binding to pre-existing filaments and inducing branching. Actin branching is critical for the formation of lamellipodia (Pantaloni, D. et al., Nat Cell Biol 2, 385-91 (2000)) and p34 appears critical for this activity (Bailly, M. et al., Curr Biol 11, 620-5 (2001)). WASP and its relative, N-WASP, act with other proteins, such as the WASP-interacting protein (WIP) and the WASP-interacting SH3 protein (WISH), respectively, that modulate their activities in cells (Takenawa, T. et al., J Cell Sci 114, 1801-9 (2001); Martinez-Quiles, N. et al., Nat Cell Biol 3, 484-91 (2001); Fukuoka, M. et al., J Cell Biol 152, 471-82 (2001)).